Methods of eliminating spores

ABSTRACT

Methods of eliminating spores of spore forming bacteria from the genus  Clostridium  on a surface comprise contacting the surface with an effective amount of an acidic aqueous composition having an acidic pH of from about 1 to about 4 and comprising from about 100 to about 2000 ppm dissolved chlorine dioxide, and a surfactant system having wetting effect and a spore solubilising effect. The surfactant system comprises at least two hydrocarbon ionic surfactants stable for oxidation at the acidic pH, of which at least two surfactants have a difference in hydrocarbon chain length of at least four carbon atoms.

RELATED APPLICATION

This application is a divisional application of U.S. application Ser.No. 14/183,314 filed Feb. 18, 2014 and claims priority thereof under 35U.S.C. 120.

FIELD OF THE INVENTION

The present invention is directed to new methods and compositionsemploying chlorine dioxide adapted to be effective for inhibiting sporeforms of bacteria.

BACKGROUND OF THE INVENTION

Chlorine dioxide is a well-known disinfecting agent and has been usedfor water cleaning and water processing since the 1950s. Chlorinedioxide is notorious labile compound as it is a powerful oxidizingagent. As chlorine dioxide generally is regarded as unstable duringstorage in aqueous solutions, numerous technical disclosures advicesystems and devices how to produce chlorine dioxide at the point of use.One commonly employed is to combine a solution of sodium chlorite with astrong acidic solution, for example comprising hydrochloric acid inorder to produce immediately applicable chlorine dioxide formulations.WO 2007/079287 (CDG Technology Inc.) discloses methodologies tostabilize dilute chlorine dioxide solutions in order to prolong productshelf life. WO2009/058530 (Ecolab) discloses compositions exhibiting lowconcentrations of chlorine dioxide together with an anionic surfactant,which demonstrate efficacy for inactivating coccidian parasites.

US Patent application 2009/0028965 (Clinimax Limited) discloses amulti-part composition capable of generating a chlorine dioxide solutionat point use. The resulting compositions comprise, besides dilutechlorine dioxide, sodium lauryl sulphate as a detergent and hold a pH atabout 5.0. Even if this document allegedly exhibits promising results,related products and other chlorine dioxide releasing products appear tohave limited effects on spores of Clostridium difficile when tested inhealth care environmental sites, see SD Goldenberg et al. in Journal ofHospital Infection, 2012, Vol. 82, pp. 64-67. The problem is alsoconfirmed by S. Ali et al in Journal of Hospital Infection, 2011 Vol.79, pp 78-79, wherein it is reported that commercial sporicidalpreparations fail to satisfyingly eliminate spores on surfaces even ifthey apparently meet sporicidal requirements when tested on sporesuspension tubes. It is evident that there both is a lack of effectivedisinfectants and also test methodologies to ascertain effectivenessagainst surface associated spores from Clostridium difficile and similarpathologic spore forming organisms.

It would therefore be strongly desirable to provide suitably stable anddilute chlorine dioxide solutions adapted to effectively inhibit alsospores of virulent spore forms of virulent bacteria, such as Clostridiumdifficile on surfaces in hospital environments. Advantageously, suchdisinfective solution should be safe to store and handle, while beingconvenient to administer at exposed surfaces, also in populated hospitalenvironments without needing any relocation to specifically designatedsanitization areas, while counteracting corrosiveness and consideringgeneral hygienic limits for chlorine dioxide.

DESCRIPTION OF THE INVENTION

In most general terms the present invention is directed to acidicaqueous compositions for elimination of spores of spore formingbacteria, comprising from about 100 to about 2000 ppm dissolved chlorinedioxide, a surfactant system having both a wetting effect and a sporesolubilising effect. Other representative examples of concentrationranges of chlorine dioxide for the compositions of the invention areabout 100 to about 1600 ppm; about 100 to about 1000 ppm; and about 500to about 1000 ppm. Examples of suitable concentrations of chlorinedioxide in composition according to the invention are about 100 ppm,about 200 ppm about 300 ppm, about 400 ppm, about 500 ppm, about 600ppm, about 700 ppm, about 800 ppm, about 900 ppm, about 1000 ppm, about1100 ppm about 1200 ppm, about 1300 ppm about 1400 ppm, about 1500 ppmand about 1600 ppm, In this respect the term “about” represents the factsince the concentration of chlorine dioxide in the composition productsmay slightly decay from their manufacturing to their use. Thecompositions generally have a pH of about 1 to about 4. The compositionsare inventively adapted to eliminate spores of bacteria in particularbacteria species of the genus Clostridium, such as Clostridium difficileon surfaces on objects made of glass, wood, metal and various plasticmaterials in particular of surfaces in hospital environments. Thecompositions are especially adapted to eliminate spores which adhere tocavities, or microcavities or other irregular surface structures thatare difficult to reach with conventional disinfectants. The compositionsare further adapted to minimize environmental impact during thedisinfection process, while maximizing their disinfection capacity evenat short contact times below 10 minutes. The term eliminating does inthe context of the present invention mean that the spores are destroyedand do not survive to replicate.

For these purpose, the compositions comprise an effective amount ofchlorine dioxide with a capacity of exerting a suitably powerfuloxidative attack on the spore and system of surfactants comprisingsurfactants with different complementary effects by supporting thedisinfecting capacity of chlorine oxide, also in comparatively lowconcentrations. Accordingly, the inventive compositions comprise atleast one surfactant with a capacity to contribute to a wetting effectof the composition and at least one surfactant that contribute to aspore solubilising effect.

In this context a “wetting effect” means that the compositions arecapable is readily applicable to administer to the surface and todistribute to the surface to form cohesive films. In the meaning of thepresent invention, “wetting effect” contributes to that the chlorinedioxide composition is suitably distributed to cavities of the surfacesin order to enable suitable elimination contact between dissolvedchlorine dioxide and spores.

Also, in all contexts of the invention “solubilising effect” means thatthe compositions are capable reduce or eliminate the adhesion of thespores to the surface. The “solubilising effect” is related to effect of“general detergency” of the compositions which would include removingthe spores from surface, contributing to support contact with chlorinedioxide and make the spores liable for a chemical oxidative attack.

Further in this context, the terms that a surfactant contributes to“wetting effect” does not exclude that it has also a certain“solubilising effect and vice versa. The intended meaning is that atleast one surfactant have more pronounced contribution to a wettingeffect than a solubilising effect and that at least on surfactant has amore pronounced solubilising effect than a wetting effect.

Further in general terms, in order to meet the requirements ofeffectively provide a wetting effect and a solubilising effect in thespecific recited chlorine dioxide containing compositions, thesurfactant system comprises two or more hydrocarbon ionic surfactantsstable for oxidation at an acidic pH of about 1 to about 4, of which atleast two surfactants have a difference in hydrocarbon chain length. Anexample of a suitable difference is at least four carbon atoms.

“Hydrocarbon ionic surfactants” is defined in this context that they areionic at an acidic pH and that they comprise a straight or branchedhydrocarbon chain. The hydrocarbon chain may be saturated or unsaturatedand thereby termed straight or branched alkyl, alkenyl or alkynyl. Thehydrocarbon chain may further be substituted with groups that contributeto or improve any surface activity or induce stability. The hydrocarbonchain can further be interrupted with one or more heteroatoms, asexemplified by alkoxy chains. In the context of ionic surfactants, theskilled artisan will have a clear understanding of what signifies ahydrocarbon chain and that different brands of surfactants basedhydrocarbon ionic surfactants can vary the chain length, when based onthe same chemical structures providing the ionic groups.

In this context “stability” means that the surfactants are notsubstantially degraded in an acidic pH of about 1-4 and that they arenot liable for oxidation by chlorine dioxide, i.e. “chlorine dioxidestable”. In other terms stability means substantially stable fromchemical degradation also during shipping and storage, but also from anyform of physical destabilization in the solution, such as precipitation,clouding or any other phenomena that affects surfactant functionality orany general visual appearance of the compositions.

In a general aspect, the compositions with chlorine dioxide according tothe invention includes a surfactant system comprising two or morehydrocarbon ionic surfactants stable for oxidation at an acidic pH,wherein at least two surfactants having hydrocarbon chains withdifferent chain lengths in order to provide the surfactant system withthe mentioned, desirable characteristics.

In one aspect, the compositions according to the invention pertains toan acidic aqueous composition having a pH of from about 1 to about 4,for elimination of spores of spore forming bacteria, comprising fromabout 100 to about 2000 ppm dissolved chlorine dioxide, a surfactantsystem having wetting effect and a spore solubilising effect, whereinthe surfactant system comprises at least two hydrocarbon ionicsurfactants stable for oxidation at a pH of about 1 to about 4, of whichsaid at least two surfactants have a difference in hydrocarbon chainlength of at least four carbon atoms.

In one aspect, the composition two or more surfactants are present inamount of from about 0.01 to about 2% (v/v), exemplified with to 0.02 toabout 2% (v/v) about 0.1 to about 1% (v/v).

In one aspect, two or more surfactants are cationic surfactants at a pHin the range of from about 1 to about 4.

In one aspect, the compositions according to the invention comprise twoor more cationic surfactants include amine oxides stable at the low pHand in presence of chlorine dioxide. Amine oxides suitable for use inthe present invention include alkyl di(lower alkyl) amine oxides inwhich the alkyl groups can have 6-20 carbon atoms, preferably, 8-18carbon atoms and can have a straight or branched chain that is saturatedor unsaturated, The lower alkyl groups have from 1 to 7 carbon atoms.Examples include C-8 and C-12 amine oxides and in one aspect that thecompositions include one surfactant that is a C-8 amine oxide and onesurfactant that is a C-12 amine oxide. Examples of such surfactantsinclude octyldimethylamine oxide and lauryldimethylamine oxide.

Alternative amine oxides include alkyl (dihydroxy lower alkyl) amineoxides, wherein the alkyl groups can have 6-20 carbon atoms, preferably,8-18 carbon atoms and can have a straight or branched chain that issaturated or unsaturated. The lower alkyl groups have from 1 to 7 carbonatoms.

Still alternative amine oxides include alkylmorpholine oxides havingalkyl groups with 6-20, preferably 6 to 12 carbon atoms.

In one aspect of the invention, the compositions comprise from about 100to about 2000 ppm chlorine dioxide, from about 0.1 to about 2 (v/v) % oftwo cationic surfactants having hydrocarbon chains with different chainlengths in order to establish both a wetting effect and a solubilisingeffect, as discussed above, the cationic surfactants can be present inthe composition in an equal amount or an about equal amount. Thecationic surfactants can differ in chain length with four carbon atoms,or more.

In one aspect, the composition comprise from about 100 to about 1600 ppmchlorine dioxide, and the amount of surfactants are about 0.2% (v/v)which comprise essentially equal amounts of octyldimethylamine oxide andlauryldimethylamine oxide.

In one aspect of the invention, the composition includes the two or moresurfactants which are anionic surfactants, stable at a pH in the rangeof from about 1 to about 4. The anionic surfactants are selected so asto contribute both to a wetting effect and a solubilising effect.

In one aspect of the invention, the compositions comprise from about 100to about 1600 ppm chlorine dioxide, from about 0.1 to about 1 (v/v) % oftwo anionic surfactants having hydrocarbon chains with different chainlengths in order to establish both a wetting effect and a solubilisingeffect, as discussed above, the anionic surfactants can be present inthe composition in an equal amount or an about equal amount. The anionicsurfactants can differ in chain length with four carbon atoms, or more.

In one aspect of the invention, the two more anionic surfactants areselected from group consisting of soluble salts of alkyl sulphates,alkyl sulphonates, alkyl aryl sulphonates (alkylbenzene sulphonates),and aryl sulphonates having from 6 to 25 carbon atoms in the alkylchains, or between 8 to 19 carbon atoms in the alkyl chains.

In one aspect, the anionic surfactants are sodium salts ofalkyl-diphenyloxide-disulphonates or alkyl-phenyl/diphenyl-sulphonateswith 6 to 19 carbon atoms in the alkyl chains. One example of anionicsurfactant is alkyl diphenyloxide disulphonate and/oralkyl(sulphophenoxy)benzene sulphonate. One example isdecyl(sulfophenoxy)-disodium salt as included in the product sold by DowChemical Company under the tradename Dowfax 3B2. One example is mono-and dihexadecyl disulphonted diphenyl oxide disodium salt as included inthe product sold by Dow Chemical Company under the tradename Dowfax8390.

In one aspect the invention relates to a method of elimination thespores of spore forming bacteria on a surface by contacting the surfacewith a composition comprising chlorine dioxide in an effective amountand two more surface active agents in a surfactant system, wherein atleast one surface active agent contributes to a wetting effect in orderto safely and cohesively distribute the composition to all parts of asurface, while at least one surface active agent shall contribute tosolubilising effect of the spores in the meaning directly or indirectlyaffect the spore surfaces in order make the spores more liable for theeffects of the chlorine dioxide. In this context an effective amountmeans any concentration or concentration range referred to in thisspecification. Also in this context, the at least two surface activeagents can be selected as outlined and used with any exemplifiedcomposition in the previous parts of this specification. In particular,at least two surfactants shall have significant difference inhydrocarbon chain length which means a difference of from 2 to 12 carbongroups. An exemplifying difference is 4 carbon groups. Further in thiscontext, contacting a surface includes any administration or applicationof a composition according to the invention to the surface in order toor with the purpose to sufficiently cover a surface contaminated, orregarded to be at risk to be contaminated with spores. An especiallypreferred usefulness of the method is the elimination of spores from thegenus Clostridium, especially Clostridium difficile.

In one aspect of the described methods, the elimination of spores ofspore forming bacteria on a surface are directed to contacting thesurface a sufficient amount of any composition as previously disclosedduring a limited contact time, such as 10 minutes or less, or a contacttime such as 9 minutes, 8 minutes 7 minutes, 6 minutes, 5 minutes, 4minutes, 3 minutes, 2 minutes, 1 minute or less such as 30 seconds orless.

Without being bound to any particular theory, it is apparent that thepresent invention comprising chlorine dioxide and in combination withthe described systems of different surfactants with complementaryeffects appears necessary for elimination of spores associated withsurfaces. In addition to an adequate wetting effect for distributing thecomposition to all parts of the surfaces, a detergency effect isobtained by which adhesion between the spores and surfaces is abolished.In addition, the compositions according to the invention appear to havea solubilising effect directly on the spore surface which results inloss of integrity of the spores and may render them susceptible for andenhance the oxidative attack of chlorine dioxide.

In the following exemplifying part tests are outlined with compositionsand methods of the invention that verify the efficacy to eliminatespores on surfaces at very rigorous conditions at levels ofcontamination designed to exceed conditions normally encountered inclinical environments even during epidemic events.

DETAILED AND EXEMPLIFYING DESCRIPTION Example 1 Manufacturing ofChlorine Dioxide Compositions

Chlorine dioxide cannot be conventionally stored as a gas due to itsexplosiveness, but is generally highly soluble in water. Chlorinedioxide is however extremely volatile and it has remained a problem tomanufacture, ship and store concentrated chlorine dioxide solutionswithout significant losses. In the context of the present inventionaqueous chlorine dioxide compositions can be produced according todifferent protocols as outline in EPA Guidelines Manual AlternativeDisinfectants and Oxidant, April 1999, Chapter 4. Chlorine Dioxide,pages 4-1 to 4-41.

For the purpose of producing compositions having from 100 to 2000 ppmdissolved chlorine dioxide of the present invention, chlorine dioxide isgenerated by mixing 9% (w/v) HCl and 7% (w/v) NaClO₂ (sodium chlorite)which rapidly reacts to form chlorine dioxide ClO₂ and NaCl in a highlyacidic environment.

A prototype stock solution of 5 litres comprising approx. 1600 ppmchlorine dioxide was prepared by mixing 300 ml 9% (w/v) HCl and 300 ml7% (w/v) NaClO₂ (sodium chlorite) diluted with 4.4 litres highlypurified water (Millipore Milli-Q).

In order to balance out the decay of concentration between the moment ofproduction and moment of surface tests according to following examples,a stock solution was made with an intended surplus of chlorine dioxideof about 10-20%. For this reason, the concentration values of theexemplified solutions during the tests raging from 100-1600 ppm shall beregarded as nominal and may vary up to 20%, such as 10-20% from theirindicated concentration values.

Example 2A

A solution for testing was produced from the stock solution by mixing

1800 mL stock solution.

1.8 mL Macat® AO-8 (approx. 30% Octyl Dimethylamine Oxide)

1.8 mL Macat® AO-12 (approx. 30% Lauryl Dimethylamine Oxide) to a testsolution of a pH of approximately 1 with approximately 1600 ppm chlorinedioxide, including about 0.1% (v/v) AO-8 and about 0.1% (v/v) AO-12.

Example 2B

A number of test solutions were generated from the stock solutionproduced according to Example 1 with the cationic surfactants of Example2 with different levels of chlorine dioxide.

Aliquotes from the solution in example 2 was further diluted 2, 4, 8, 16times with MilliQ-water containing about 0.1% (v/v) AO-8 and 0.1% (v/v)AO12. The final set of test solutions contained 1600, 800, 400, 200 and100 ppm ClO₂ with a total amount of surfactant of 0.2% (v/v).

Example 3

A second solution for testing was produced from the stock solution bymixing 1000 ml stock solution with 1 mL DOWFAX® 3B2 (approx 38%Benzenesulfonic acid, decyl(sulfophenoxy)-, disodium salt and approx 8%Benzenesulfonic acid, oxybis(decyl)-, disodium salt and 1 mL DOWFAX®8390 (38.5% % Mono- and dihexadecyl disulphonted diphenyl oxide disodiumsalt) to a test solution of a pH of approximately 1, includingapproximately 1600 ppm chlorine dioxide.

Example 4

In this example, a methodology is outlined to investigate ifcompositions according to present invention exhibit an eliminationcapacity for spore forming bacteria on surfaces. The example alsocompares with conventional anti-bacterial agents and confirms theirrelative inefficacy.

Elimination of Spores of Clostridium difficile on Surfaces

Methods and Materials

Spore Suspension

C difficile spores PCR Ribotype 023 (ECDC/Cardiff nomenclature) weretransferred from a frozen sample to plates with Fastidious Anaerobe Agar(FAAAP), cultured under anaerobe conditions for 2 days. The cultureswere transferred to ambient conditions and colonies of C difficile wereadmitted to sporulate. The spores were harvested and dissolved insterile water (reverse osmosis (RO)), subjected to alcohol shock (70%),washed, centrifugated and collected as a suspension of spores. TheSpores were counted in a Barker chamber to 2.4×10⁸ spores (i.e. sporeson the tested surfaces) comparing with approximately 80% spores in thesuspension.

Agar Plates

In the experiment Fastidious Anaerobe Agar (FAAAP) was with addedtaurocholate (1 g/l), except from control of the filtrate, where agarplates selective for C. difficile was used.

Preparation of Surfaces

In the preparation 0.1 ml of the spore suspension was transferred tomicroscopy slides, sterile plates of brass or copper. When the sporesuspensions have dried, 0.2 ml of the following solutions were added:

A solution made in accordance with Example 2A

Virkon 1%

Ethanol 70%

Sterile RO-water

After 10 minutes exposure to the solutions, the Glasses and the plateswere each transferred to a 500 ml Glass flask to dilute and interruptdisinfection. The flasks were rotated at 200 rpm for 20 minutes.

Culturing

From each of the rotated flasks 0.1 ml was transferred to agar plates(Culture 1). The samples were diluted in two steps 1/10 in tubes. Fromeach tube, 0.1 ml was transferred to agar plates (Cultures 2 and 3).

The solutions in the flasks were filtered and rinsed with 3×100 mlbuffered peptone water (PENAL). The filtrations were performed in twosteps. At first, 1 ml was filtered, then remaining volume of solution inthe flasks (approx. 249 ml). The filters were cultured on agar plates(Cultures 4 and 5).

At two times, 40 ml samples were taken from the filtrates. These werecentrifugated and the supernatant was discarded, the pellet re-suspendedin 0.1 ml 0.85% NaCl solution. The entire volume was transferred to agarplates.

In order to assess if viable spore remain on the surfaces of Glasses andthe, they were smeared with moist cotton tip (all smeared in accordancewith a “double-S” shape). The tip was cultured in 2 ml pre-reducedPY-broth which was diluted 1/10 in several repeated steps and culturedon agar plates (0.2 ml/plate), (Cultures 6 to 9)

All culture plates were cultured under anaerobe conditions at 36° C. for2 days.

The results are demonstrated in Table 1, below.

In Table 1, results are demonstrated for Culture 1 to 9 for the threedifferent surfaces (Glass, brass, copper) with the four different testsolutions. The results within brackets refer to total amount of CFUs(colony forming units) in the flasks, i.e. the amount of spores thathave survived treatment with the tests solutions and have been displacedfrom the surfaces during rotation of the flasks. Table 1 alsodemonstrates the amount of CFU in the undiluted PY-broth referring theamount of spores surviving treatment of the test solution, but haveremained attached to the surface during rotation of flasks.

Table 1 demonstrates that the test solution referring to Example 2 hasexcellent capacity to eliminate spores of C difficile. It is alsosignificant that the test solution according to Example 2A has capacityto eliminate spores which adhere to a surface and thereby would bedifficult to eliminate or less susceptible for conventional eliminationagents.

In addition to the results of Table 1, it was confirmed that no growthwas found in the cultures from the samples taken from the filtrate.

TABLE 1 Culture 1 Culture 2 Culture 3 Culture 4 Culture 5 Culture 6Culture 7 Culture 8 Culture 9 0.1 mL From Dilution Dilution FiltrationFiltration PY-broth PY-broth PY-broth PY-broth bottle 10⁻¹ 10⁻² 1 mL 249mL undiluted 10⁻² 10⁻⁴ 10⁻⁶ RO-water overgrown 527 53 overgrownovergrown overgrown overgrown 24 3 Glass (1.3 × 10⁷) (2.4 × 10⁶)RO-water overgrown 254 23 overgrown overgrown overgrown overgrown 45 1Brass (6.4 × 10⁶) (4.5 × 10⁶) RO-water overgrown 155 14 overgrownovergrown overgrown overgrown 62 1 Copper (3.9 × 10⁶) (6.2 × 10⁶) Virkon1% overgrown 189 10 overgrown overgrown overgrown overgrown 94 0 Glass(4.7 × 10⁶) (9.4 × 10⁶) Virkon 1% 461  32  2 overgrown overgrownovergrown overgrown 13 0 Brass (1.1 × 10⁶) (1.3 × 10⁶) Virkon 1%overgrown  68  2 overgrown overgrown overgrown overgrown 15 0 Copper(1.7 × 10⁶) (1.5 × 10⁶) Example 2A  0  0  0 0  4 0 0  0 0 Glass EXAMPLE 0  0  0 0 84 0 0  0 0 2A Brass EXAMPLE  0  0  0 0  9 0 0  0 0 2A Copper70 % Ethanol overgrown 517 46 overgrown overgrown overgrown overgrown 391 Glass (1.2 × 0⁷) (3.9 × 10⁶) 70 % overgrown  55  5 overgrown overgrownovergrown overgrown 74 0 Ethanol/Brass (1.4 × 10⁶) (7.4 × 10⁶) 70 %Ethanol 429  40  1 overgrown overgrown overgrown overgrown 79 0 Copper(1.0 × 10⁶) (7.9 × 10⁶)

Example 5

Sterile microscopy slides (glass surfaces) with spore suspension wereprepared in accordance with Example 4.

The solutions prepared in Example 2B containing 1600, 800, 400, 200 and100 ppm ClO₂ with a total amount of surfactant of 0.2% (v/v) wereapplied to the glass surfaces in accordance with Example 4.

The glass slides were treated in the same way as in Example 4 andsamples were taken and cultured in the same way as in Example 4.

The results are demonstrated in Table 2 for the solutions comprising1600, 800, 400, 200 and 100 ppm ClO₂. The results are presented in thesame way as in Table 1.

It is evident that compositions according to the invention having so lowconcentrations of chlorine dioxide as 100 ppm have an improved efficacyof eliminate spores of C difficile, compared to conventional agents,such as ethanol and Virkon. The results also indicate that compositionsaccording to the invention have capacity to exert its effect ondifferent types of surfaces.

In addition to the results of Table 1, it was confirmed that no growthwas found in the cultures from the samples taken from the filtrate.

TABLE 2 0.1 mL From Dilution Dilution Filtration Filtration PY-brothPY-broth PY-broth PY-broth bottle 10⁻¹ 10⁻² 1 mL 249 mL undiluted 10⁻²10⁻⁴ 10⁻⁶ RO-water I overgrown overgrown 170 overgrown overgrownovergrown overgrown 200 4 (4.2 × 10⁷) (2.0 × 10⁷) RO-water II overgrownovergrown 107 overgrown overgrown overgrown overgrown 177 0 (2.7 × 10⁷(1.8 × 10⁷)  100 ppm 130 8  1 overgrown overgrown overgrown overgrown159 1  (3.2 × 10⁵) (1.6 × 10⁷)  200 ppm  57 3 overgrown overgrownovergrown overgrown overgrown  67 0 (1.42 × 10⁵) (6.7 × 10⁶)  400 ppm 18 0  0 108 overgrown overgrown overgrown  27 0 (2.7 × 10⁷) (2.7 × 10³) 800 ppm 0 0  0  9 1000 16 0  0 0 (2.2 × 10³) (1.6 × 10³) 1600 ppm 0 0 0  0  38  0 0  0 0 (3.8 × 10³)

Example 6

Example 6 was designed in order to evaluate different types andcombinations of surface active agents in combination with chlorine oxidefor elimination of spores of C Difficile on surfaces. The following testsolutions were used:

Sterile RO-Water

Ethanol 70%

Solution LC1 comprising: 1600 ppm chlorine dioxide

Solution LC1_12 comprising: 1600 ppm chlorine dioxide with 0.2% (v/v)RD1 and RD2

Solution LC1_34 comprising: 1600 ppm chlorine dioxide with 0.2% (v/v)RD3 and RD4

Solution LC2 comprising 800 ppm chlorine dioxide

Solution LC2_1 comprising 800 ppm chlorine dioxide with 0.2% (v/v) RD1

Solution LC2_2 comprising 800 ppm chlorine dioxide with 0.2% (v/v) RD2

Solution LC2_3 comprising 800 ppm chlorine dioxide with 0.2% (v/v) RD3

Solution LC2_4 comprising 800 ppm chlorine dioxide with 0.2% (v/v) RD4

Solution LC2_12 comprising: 800 ppm chlorine dioxide with 0.2% (v/v) RD1and RD2

Solution LC2_34 comprising: 800 ppm chlorine dioxide with 0.2% (v/v) RD3and RD4

The solutions were prepared in accordance with Examples 2, 2A and 3. RD1and RD2 signify the anionic surfactants of Example 3 branded DOWFAX 3B2and DOWFAX 8390, respectively. RD3 and RD4 signify the cationicsurfactants of Example 2A branded AO-8 Macat and AO-12 Macat.

A spore suspension was generated and counted in accordance with Example4:

Ribotype 25; 8.0×10⁹ spores/mL (8.0×10⁸ spores on the glass surface)approximately 80% spores in the suspension.

The glass surfaces were prepared by transferring 0.1 ml of the sporesolution to microscopy slides. After the suspension has dried, 0.2 ml ofthe test solutions were added to the slides for application timesbetween 1 and 10 minutes. Subsequently, the glass surfaces were eachplaced in a 500 ml glass flask with 250 ml 0.85% NaCl solution androtated at 200 rpm for 20 minutes.

The results of the cultures in observed CFUs are shown in Table 4, alsodemonstrating with brackets the number of CFUs in the flask and thenumber of CFUs in the undiluted PY-broth, representing the number ofsurviving spores detached from the surface and the number of sporessurvived not detached from the surface during the rotation,respectively.

In addition to the results of Table 3, it was confirmed that no growthwas found in the cultures from the samples taken from the filtrate.Table 3 demonstrates that the spore eliminating efficacy was improvedwhen combing two surfactants with different complementarycharacteristics when compared to solution comprising a singlesurfactant.

TABLE 3 0.1 mL 0.1 mL 0.1 mL PY-broth PY-broth Filtration FiltrationFrom Diluted Diluted PY-broth Diluted Diluted Filtration 249 mL 1 mLbottle 10⁻¹ 10⁻² Undiluted 10⁻² 10⁻⁴ 249 mL RO- overgrown overgrownovergrown overgrown 292 overgrown overgrown 13 0 water (7.3 × 10⁷) (1.3× 10⁶) 70% overgrown overgrown overgrown overgrown 240 overgrownovergrown 86 0 ethanol (6.0 × 10⁷) (8.6 × 10⁶) LC1 150  0  0 0  0  0 0 0 0 (150) LC1_12  55  0  0 0  0  0 0  0 0  (55) LC1_34  0  0  0 0  0  00  0 0 LC2 overgrown 200 36 2  0  28 0  0 0 (5.0 × 10⁴) (280) LC2_1overgrown  85 13 0  0 176 3  0 0 (2.1 × 10⁴) (1.8 × 103) LC2_2 overgrown101 15 2  0  2 0  0 0 (2.5 × 10⁴)  (20) LC2_3 overgrown  49  5 0  0  110  0 0 (1.2 × 10⁴) (110) LC2_4 overgrown  13  3 0  0  36 0  0 0 (3.2 ×10³) (360) LC2_12 overgrown 120 17 1  0  3 0  0 0 (3.0 × 10⁴) LC2_34overgrown  8  1 0  0  0 0  0 0 (2.0 × 10³)

Example 7

Example 7 outlines a test with different contact for chlorine dioxidesolution according to the invention. A spore suspension was generatedand counted in accordance with Example 4:

Ribotype 25; 9.6×10⁸ spores/mL (9.6×10⁷ spores on the glass surface)approximately 80% spores in the suspension.

The glass surfaces were prepared by transferring 0.1 ml of the sporesolution to microscopy slides. After the suspension has dried, 0.2 ml ofthe test solutions were added to the slides for application timesbetween 1 and 10 minutes. Subsequently, the glass surfaces were eachplaced in a 500 ml glass flask with 250 ml 0.85% NaCl solution androtated at 200 rpm for 20 minutes.

The test solutions were:

Sterile RO-water 10 min

Ethanol 70% 10 min

Chlorine dioxide 1600 1 min

Chlorine dioxide 1600 2 min

Chlorine dioxide 1600 3 min

Chlorine dioxide 1600 4 min

Chlorine dioxide 1600 5 min

Chlorine dioxide 1600 6 min

Chlorine dioxide 1600 7 min

Chlorine dioxide 1600 8 min

Chlorine dioxide 1600 9 min

Chlorine dioxide 1600 10 min

The test solution comprising chlorine dioxide was prepared in accordancewith Example 2B, comprising 1600 ppm chlorine dioxide together with 0.2%(v/v) cationic surfactant comprising equal amounts of octyldimethylamine oxide and lauryl dimethylamine oxide). After rotation, thecontents of the flasks were treated and cultured with procedures andagar plates in accordance with Example 4.

The results of the cultures in observed CFUs are shown in Table 4, alsodemonstrating with brackets the number of CFUs in the flask and thenumber of CFUs in the undiluted PY-broth, representing the number ofsurviving spores detached from the surface and the number of sporessurvived not detached from the surface during the rotation,respectively.

In addition to the results of Table 4, it was confirmed that no growthwas found in the cultures from the samples taken from the filtrate.

The results of Table 4 indicate that the test solution is highlyefficient for the highly spore contaminated surfaces also at very lowcontact times. Also at contact times as low as 1 minute, or less testsolution provides suitable efficacy to admit chlorine dioxide to a exertspore eliminating effect.

A repeated test based on a spore suspension (Ribotype 25: 4.8×10⁸spores/mL (. 4.8×10⁷ on the glass surface) about 80% spores insuspension) shows efficacy with as low contact time as 15 seconds withthe same test solution.

TABLE 4 Number Number Number Number Number cfu × 10 × cfu × 100 × cfu ×1000× Number Number Number cfu cfu × 250 250 250 250 cfu × 10 cfu × 10³cfu × 10⁵ 0.1 mL 0.1 mL 0.1 mL PY-broth PY-broth Filtration FiltrationFrom Diluted Diluted PY-broth Diluted Diluted 249 mL 1 mL bottle 10⁻¹10⁻² Undiluted 10⁻² 10⁻⁴ RO-water overgrown overgrown overgrownovergrown 171 overgrown overgrown 9 (4.3 × 10⁷) (9.0 × 10⁵) 70%overgrown overgrown overgrown overgrown 103 overgrown overgrown 67ethanol (2.6 × 10⁷) (6.7 × 10⁶) ClO₂  27 1 0 0  0 0 0 0 1 min ClO₂  23 00 0  0 0 0 0 2 min ClO₂ 100 0 0 0  0 0 0 0 3 min ClO₂  83 0 0 0  0 0 0 04 min ClO₂  95 0 0 0  0 0 0 0 5 min ClO₂  52 0 0 0  0 0 0 0 6 min ClO₂ 21 0 0 0  0 0 0 0 7 min ClO₂ 105 0 0 0  0 0 0 0 8 min ClO₂ 153 3 0 0  00 0 0 9 min ClO₂  50 1 0 0  0 0 0 0 10 min

Example 8

Example 9 outlines a test with chlorine dioxide solutions of 800 and1600 ppm having different concentration of surfactants made inaccordance with Example 2A. A spore suspension was generated and countedin accordance with Example 4:

Ribotype 25; 8.0×10⁸ spores/mL (8.0×10⁷ spores on the glass surface)approximately 80% spores in the suspension.

The glass surfaces were prepared by transferring 0.1 ml of the sporesolution to microscopy slides. After the suspension has dried, 0.2 ml ofthe test solutions were added to the slides for an application time of 2minutes. Subsequently, the glass surfaces were each placed in a 500 mlglass flask with 250 ml 0.85% NaCl solution and rotated at 200 rpm for20 minutes.

The test solutions were:

Sterile RO-water

Ethanol 70%

LC1A_2 comprising 1600 ppm chlorine dioxide

LC1B_2 comprising 1600 ppm chlorine dioxide with 2% (v/v) RD3 and RD4

LC1C_2 comprising 1600 ppm chlorine dioxide with 0.02% (v/v) RD3 and RD4

LC1E_2 comprising 1600 ppm chlorine dioxide with 0.2% (v/v) RD3 and RD4

LC2A_2 comprising 800 ppm chlorine dioxide

LC2B_2 comprising 800 ppm chlorine dioxide with 2% (v/v) RD3 and RD4

LC2C_2 comprising 800 ppm chlorine dioxide with 0.02% (v/v) RD3 and RD4

LC2E_2 comprising 800 ppm chlorine dioxide with 0.2% (v/v) RD3 and RD4

RD3 and R4 signify, the cationic surfactants of Example 2A Macat® AO-8and Macat® AO-12.

After rotation, the contents of the flasks were treated and culturedwith procedures and agar plates in accordance with Example 4.

The results of the cultures in observed CFUs are shown in Table 5, alsodemonstrating with brackets the number of CFUs in the flask and thenumber of CFUs in the undiluted PY-broth, representing the number ofsurviving spores detached from the surface and the number of sporessurvived not detached from the surface during the rotation,respectively.

In addition to the results of Table 5, it was confirmed that no growthwas found in the cultures from the samples taken from the filtrate.

Table 5 demonstrates that a chlorine dioxide solution concentration of0.2% (v/v) has the more advantageous performance to eliminate spores onsurfaces, compared both to higher and lower concentrations of surfactantat both tested strengths of chlorine dioxide (800 and 1600 ppm). Theresults also confirm the efficacy of the present surfactant to assistchlorine dioxide to exert its eliminating effect.

TABLE 5 Number Number Number Number Number cfu x 10 × cfu × 100 × cfu ×1000 × Number Number Number cfu cfu × 250 250 250 250 cfu × 10 cfu × 103cfu × 105 0.1 mL 0.1 mL 0.1 mL PY-broth PY-broth Filtration FiltrationFrom Diluted Diluted PY-broth Diluted Diluted Filtration 249 mL 1 mLbottle 10⁻¹ 10⁻² Undiluted 10⁻² 10⁻⁴ 249 mL RO- overgrown overgrownovergrown overgrown 97 overgrown  61  0 water (2.4 × 10⁷) (6.1 × 10⁴)70% overgrown overgrown overgrown overgrown 81 overgrown overgrown 28ethanol (2.0 × 10⁷) (2.8 × 10⁶) LC1A_2 overgrown 98  13  2  0 overgrown 23  0 (2.4 × 10⁴) (2.3 × 10⁴) LC1B_2 overgrown 74  6  0  0 overgrown 28  0 (1.8 × 10⁴) (2.8 × 10⁴) LC1C_2 overgrown 20  4  0  0 overgrown  8 0 (5.0 × 10³) (8.0 × 10³) LC1E_2 overgrown  8  0  0  0 overgrown  10  0(2.0 × 10³) (1.0 × 10⁴) LC2A_2 overgrown overgrown overgrown 29  0overgrown 287  4 (7.2 × 10⁵) (2.9 × 10⁵) LC2B_2 overgrown overgrown 150 9  0 overgrown 136  0 (3.8 × 10⁵) (1.4 × 10⁵) LC2C_2 overgrownovergrown overgrown 23  2 overgrown 129  1 (5.8 × 10⁵) (1.3 × 10⁵)LC2E_2 overgrown overgrown overgrown 35  3 overgrown  96  1 (8.8 × 10⁵)(9.6 × 10⁴)

1. A method of eliminating spores of spore forming bacteria from the genus Clostridium on a surface, comprising contacting the surface with an effective amount of an acidic aqueous composition having an acidic pH of from about 1 to about 4 and comprising from about 100 to about 2000 ppm dissolved chlorine dioxide, and a surfactant system having wetting effect and a spore solubilising effect, wherein the surfactant system comprises at least two hydrocarbon ionic surfactants stable for oxidation at the acidic pH, of which at least two surfactants have a difference in hydrocarbon chain length of at least four carbon atoms.
 2. The method of claim 1, wherein the two or more surfactants are present in amount of from about 0.01 to about 2% (v/v).
 3. The method of claim 1, wherein the two or more surfactants are cationic surfactants at a pH in the range of from about 1 to about
 4. 4. The method of claim 1 wherein the two or more surfactants are amine oxides having alkyl groups with 6 to 18 carbon atoms.
 5. The method of claim 4; wherein one surfactant is a C-8 amine oxide and one surfactant is a C-12 amine oxide.
 6. The method of claim 5, wherein one surfactant is octyldimethylamine oxide and one surfactant is lauryldimethylamine oxide.
 7. The method of claim 6, wherein the composition comprises about 0.2% (v/v) of the two or more surfactants and the two or more surfactants comprise essentially equal amounts of octyldimethylamine oxide and lauryldimethylamine oxide.
 8. The method of claim 1, wherein the two or more surfactants are anionic surfactants at a pH in the range of from about 1 to about
 4. 9. The method of claim 8, wherein the two more anionic surfactants are selected from the group consisting of soluble salts of alkyl sulphates, alkyl sulphonates, alkyl aryl sulphonates, and aryl sulphonates having between 7 and 25 carbon atoms.
 10. The method of claim 9, wherein the surfactants are alkyl-diphenyloxide-disulphonates with 12 to 19 carbon atoms in the alkyl chains.
 11. Method of claim 1, wherein the spore forming bacteria are Clostridium difficile. 